The present invention relates to the field of glycobiology, and specifically to glycosyltransferases, a superfamily of enzymes that are involved in synthesizing the carbohydrate moieties of glycoproteins, glycolipids and glycosaminoglycans. The present invention provides the structure-based design of novel glycosyltransferases and their biological applications.
Glycans can be classified as linear or branched sugars. The linear sugars are the glycosaminoglycans comprising polymers of sulfated disaccharide repeat units that are O-linked to a core protein, forming a proteoglycan aggregate (Raman et al. 2005). The branched glycans are found as N-linked and O-linked sugars on glycoproteins or on glycolipids (Lowe et al., 2003). These carbohydrate moieties of the linear and branched glycans are synthesized by a super family of enzymes, the glycosyltransferases (GTs), which transfer a sugar moiety from a sugar donor to an acceptor molecule. Although GTs catalyze chemically similar reactions in which a monosaccharide is transferred from an activated derivative, such as a UDP-sugar, to an acceptor, very few GTs bear similarity in primary structure.
Eukaryotic cells express several classes of oligosaccharides attached to proteins or lipids. Animal glycans can be N-linked via beta-GlcNAc to Asn (N-glycans), O-linked via -GalNAc to Ser/Thr (O-glycans), or can connect the carboxyl end of a protein to a phosphatidylinositol unit (GPI-anchors) via a common core glycan structure.
The structural information of glycosyltransferases has revealed that the specificity of the sugar donor in these enzymes is determined by a few residues in the sugar-nucleotide binding pocket of the enzyme, which is conserved among the family members from different species. This conservation has made it possible to reengineer the existing glycosyltransferases with broader sugar donor specificities. Mutation of these residues generates novel glycosyltransferases that can transfer a sugar residue with a chemically reactive functional group to N-acetylglucosamine (GlcNAc), galactose (Gal) and xylose residues of glycoproteins, glycolipids and proteoglycans (glycoconjugates). Thus, there is potential to develop mutant glycosyltransferases to produce glycoconjugates carrying sugar moieties with reactive groups that can be used in the assembly of bio-nanoparticles to develop targeted-drug delivery systems or contrast agents for medical uses.
Accordingly, methods to synthesize N-acetylglucosamine linkages have many applications in research and medicine, including in the development of pharmaceutical agents and improved vaccines that can be used to treat disease.